Method for design of substances that enhance memory and improve the quality of life

ABSTRACT

Amnestic novel peptides are disclosed. A topographical map or surface which includes binding sites for peptides having amnestic procedures and the use of such surface to identify and synthesize such peptides is also disclosed.

FIELD OF THE INVENTION

This invention relates to memory enhancement and to improvement in thequality of life for human individuals who for various reasons includingaging, disease, or injury show impairment of memory. More specifically,the invention relates to development of a topographic model on the basisof which it is possible to design and synthesize memory-enhancing andlife-quality improving substances.

DEFINITIONS AND ABBREVIATIONS

β-A4=amyloid β protein

FAAT=footshock active avoidance training

ICV=intracerebroventricular

Ala=alanine

Cys=cysteine

Asp=aspartic acid

Glu=glutamic acid

Phe=phenylalanine

Gly=glycine

His=histidine

Ile=isoleucine

Lys=lysine

Leu=leucine

Met=methionine

Asn=asparagine

Pro=proline

Gln=glutamine

Arg=arginine

Ser=serine

Thr=threonine

Val=valine

Trp=tryptophan

Tyr=tyrosine

BACKGROUND OF THE INVENTION

Immediate post-training ICY administration of a synthetic peptidehomologous to β-A4, [Gln¹¹ ]β-(1-28) [Asp Ala Glu Phe Arg His Asp SerGly Tyr Gln Val His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly SerAsn Lys (SEQ ID NO: 1)] caused amnesia for FAAT in mice indose-dependent fashion. Also amnestic were residues β-(12-28) [Val HisHis Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Ash Lys (SEQ ID NO:2)], β-(18-28) [Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys (SEQ ID NO:3)], and β-(12-20) [Val His His Gln Lys Leu Val Phe Phe (SEQ ID NO: 4)](1). These amnestic peptides have in common the tripeptidic sequence ValPhe Phe (SEQ ID NO: 5). Residue β-(1-11) [Asp Ala Glu Phe Arg His AspSer Gly Tyr Gln (SEQ ID NO: 6)], which does not contain Val Phe Phe, wasnot amnestic (this study) FAAT experiments were performed with peptidescontaining the Val Phe Phe sequence from which was derived a topographicmodel for the binding site of amnestic peptides. Since the amnesticsubstances are memory-enhancing at lower concentration than those atwhich they cause amnesia, the model can be used to deduce the structureof potential memory-enhancing peptides and non-peptidic substances.

SUMMARY OF THE INVENTION

Structure-activity study with various peptides in memory-testingparadigms in mice has made possible generation of a topographic map fora hypothetical binding surface, Z, for amnestic peptides. On the model Zbinding sites are distributed in clockwise rotation the followingdesignated loci: (1) H-bonding; (2) aromatic; (3) cationic; (4)aromatic; and (5) anionic (FIG. 1, No. 1-3). Effects on retention ofFAAT are rationalized in terms of fit to Z, making possible design ofpotential memory-modulating peptidic and non-peptidic substances. Thesimilarity in brain function in various mammals, including human beings,as well as much previous experience in the field indicates that suchsubstances will be effective memory-enhancers in men as well as mice. Inno instance known to applicant has a model such as that above beensuggested, nor have any of the memory-enhancing peptides describedherein been suggested for use in improving memory.

Administration of such substances and their congeners orally,subcutaneously, intravenously, transcutaneously, intrathecally,sublingually, rectally, or intracisternally leads to a restoration ofthe balance between excitatory and inhibitory systems in the brain, abalance which is required for optimal acquisition and retention oflearning. Their administration helps correct defects in this balancethat arise, for example, as a result of aging, infections, and injury.Administration of such substances exerts recyberneticizing effects onnervous system function.

Another aspect of this invention comprises substances that have beendesigned to mimic the actions of the active peptides but which do nothave the peptide structure and would not be subject to degradation ofpeptide-splitting enzymes in the gut or other tissues. Such organicchemical entities would have more prolonged desired effects at lowerdoses than the peptidic structures.

DESCRIPTION OF THE FIGURES

FIGS. 1-3 are computer generated models of a putative topographic site Zfor the binding of amnestic peptides. Atoms are color coded as follows:carbon--gray; oxygen--red; nitrogen--blue; hydrogen--white.

FIGS. 4-18 illustrate the fit of various peptides to the topographicsite Z of FIG. 1.

FIG. 19 depicts the decrease of amnestic potency of peptides as afunction of H-bonding capacity. The special role of C-terminal G isdiscussed in the text.

FIGS. 20A-20D depicts the dose-response curves comparing amnesticeffects of Val Phe Phe with those of the β-A4 homologue [Gln¹¹ ]β-(1-28)(SEQ ID NO:1) (FIG. 20A) and amnestic effects of Ala Val Phe Thr (SEQ IDNO: 7) with those of Val Ile Pro (SEQ ID NO: 8) (FIG. 20B). The data forcurves for [Gln¹¹ ]β-(1-28) (SEQ ID NO:1) and Val Ile Pro (SEQ ID NO:8)were taken from references 1 and 12, respectively. The dose-responsecurve for Lys Leu Phe Phe Val Ala Glu (SEQ ID NO: 9) is shown in FIG.20C. Dose-response curves for Val Phe Phe (SEQ ID NO:5), Ala Val PheThr, and Lys Leu Phe Phe Val Ala Glu (SEQ ID NO:9) are compared witheach other in FIG. 20D.

FIGS. 21A and 21B depict memory-enhancing and amnestic effects of ValPhe Phe (SEQ ID NO:5). Experimental conditions in FIG. 21A were adjustedso that vehicle controls had poor retention. In FIG. 21B the conditionswere such as to maximize retention of learning in vehicle controls.

FIGS. 22A, 22B and 22C depict a comparison of memory-enhancing potenciesof Val Phe Phe Val Val Phe (SEQ ID NO: 10), and Phe Phe Val Gly (SEQ IDNO: 11) from experiments performed to obtain poor retention in vehiclecontrols.

FIG. 23A and 23B depicts dose-dependence of memory-enhancing andamnestic effects of Phe Val under conditions of poor retention forcontrols. Comparison of effects of Val Phe (insert) with those of PheVal in the memory-enhancing dose range.

FIG. 23C depicts a log-dose plot of the same data as shown in FIG. 23Ain order to facilitate comparison with date for Gln Phe Phe Gly (SEQ IDNO: 12) in FIG. 24.

FIG. 24 depicts a log-dose plot of memory-enhancing and amnestic effectsof SEQ ID NO: 12 in experiments with mice showing poor retention withvehicle alone.

EXEMPLIFICATION OF THE INVENTION Materials and Methods

Test Animals. After 1 week in the laboratory, CD-1 male mice obtainedfrom Charles River Breeding Laboratories were caged individually 24-28hours prior to training and remained singly housed until retention wastested one week later. Animal rooms were on a 12 hour light/dark cyclewith lights going on at the hour of 0600. Median body weight was 35 g,with a range of 33-38 g. Mice were assigned randomly to groups of 15 inthe experiments reported in FIGS. 1-18 and groups of 10 for thedose-response curves (FIGS. 19 and 20) and were trained and testedbetween the hours of 0700 and 1500.

Peptides Tested. With the exception of Val Phe and Phe Val from Sigma,all peptides used in these studies were synthesized and analyzed toestablish purity by standard methods at the Beckman Research Institute.Peptides were dissolved in: (1) saline; (2) 8% vol/vol dimethylsulfoxide; (3) glacial acetic acid neutralized with HCl; or (4) NaOHneutralized with HCl. Upon testing for retention of FAAT after receivingpost-training ICV administration of 2 μl of the above vehicles, closelysimilar mean numbers of trials to criterion for the different vehicleswere obtained: (1) 6.93±0.29; (2) 6.80±0.20; (3) 6.87±0.20; and (4)6.53±0.22. Analysis of variance showing insignificant differences amongthe latter, the value for the most frequently employed (No. 2 above) wasused for comparison with groups receiving test peptides.

With exception of dose-response curves, the experiments reported belowtested whether or not there was an amnestic effect at 6.14 nmol ofpeptide per mouse. Although β-(1-18) was active at 1.5 nmol per mouse(1), the higher dose was chosen so that substances with weaker effectsmight be detected.

Apparatus, Training and Testing Procedures. The T-maze used forfootshock active avoidance training (FAAT) consisted of a black plasticalley (46 cm long) with a start box at one end and two goal boxes (17.5cm long) at the other. The start box was separated from the alley by aplastic guillotine door that prevented movement down the alley untiltraining began. The alley was 12.5 cm deep and 9.8 cm wide. Anelectrifiable stainless steel rod floor ran throughout the maze.

Mice were not permitted to explore the maze before training. A block oftraining trials began when a mouse was placed in the start box. Theguillotine door was raised and a muffled doorbell-type buzzer soundedsimultaneously; footshock was 5 seconds later through a scrambled gridfloor shocker (Colbourn Instruments, Model E13-08). The goal box firstentered during the first set of trials was designated as "incorrect",and footshock was continued until the mouse entered the other goal box,which in all subsequent trials was designated "correct" for theparticular mouse. At the end of each group of trials, the mouse wasremoved to its home cage.

As training proceeded, a mouse made one of two types of responses. Aresponse latency longer than 5 seconds was classed as an escape from thefootshock. A response latency less than or equal to 5 seconds wasconsidered an avoidance, since the mouse avoided receiving a footshock.Two exclusion criteria were applied to reduce learning variability amongmice, as follows. On the first training trials, mice with escapelatencies greater than 20 seconds were discarded. Mice not having atleast one errorless escape latency between 1.5 and 3.5 seconds ontraining trials 3 or 4 were excluded. The total exclusions were fewerthan 15%. Mice received five such training trials. One week aftertraining and post-trial administration of vehicle alone or vehiclecontaining test substance, T-maze training was resumed until each mousemade five avoidance responses in six consecutive training trials (trialsto criterion). The recall score was taken to be the percentage of testedmice remembering original training.

Well-trained animals (recall score approximately 80%) were used todetermine whether or not administered substances could cause amnesia. Inthese instances, training was performed under conditions that tend tomaximize learning (sound intensity, 65 decibels; footshock current, 0.35mA; intertrial interval, 45 seconds). In the cases in which it wasdesired to detect whether or not there was an enhancing effect onmemory, training conditions were adjusted so that the initial recallscore in vehicle controls was only approximately 20% (sound intensity,55 decibels; footshock current, 0.30 mA; intertrial interval, 30seconds).

Surgical Procedure in Preparation for Intracerebroventricular (ICV)Administration of Substances. ICV injection was the mode ofadministration of test substances because this eliminates problems ofdifferential penetration of the blood-brain barrier. The followingprocedure was performed 24-48 hours prior to training. A single hole wasdrilled through the skull over the third ventricle (-0.5 mm relative tobregma, 0.5 mm right of central suture) while the mouse, appropriatelyanesthetized with methoxyflurane, was held in a stereotaxic instrument.The third ventricle was chosen as site of ICY drug injection becauseonly a single injection is required and the drug quickly reaches limbicsystem structures, believed to be associated with memorial processes.Immediately after training, mice were anesthetized with enflurane, ashort acting anesthetic, and given an ICV injection of 2 μl of vehiclealone or test substance in vehicle delivered over a 30-second periodthrough a 31-gauge needle attached to a 10- μl syringe; the injectionwas given within 2-3 minutes after the training. Accuracy of injectionwas determined to be greater than 95% by due injection, monitoredregularly.

Statistical Treatment of Data. All of the results are expressed in termsof the mean and standard errors of the mean (SEM). Significance ofoverall effects of treatment was determined by one-way analysis ofvariance (ANOVA) run on trials to criterion. Dunnett's t-test was usedto make multiple comparison of individual test groups with controlgroups. See Bruning, J. E., et al., in Computational Handbook ofStatistics, 2d ed., Scott, Foreman and Co., Glenview, pp. 18-30,122-124, 128-130 (1977). Statistical comparison among experimentalgroups were made by Bukey's t-test. See Winer, B. J., StatisticalPrinciples in Experimentation Design, 2d ed., McGraw-Hill, New York, pp.196-210, 397-402 (1971).

RESULTS

Topography of a binding site deduced from structure-activity study ofamnestic effects. Structural examination of CPK models of the peptidestested and superimposition of appropriate configurations of the activeones led to devisal of a 2-dimensional model of the site to which all ofthe amnestic substances might bind. Subsequently, the 3-dimensionalbinding surface "Z" for the peptides of this invention was generated bysurrounding with small molecules that mimic the properties of amino acidside chains a peptide, Gln-Phe-Phe-γ-Aminobutyric acid SEQ ID NO:20 thatfits well to all of the proposed contact points on the 2-dimensionalmap. A solvent-accessible surface was generated using the algorithm ofConnolly (2). Peptides then were docked onto the surface and energyminimized to optimize contacts (Dreiding II forcefield (3)). Images werecreated with the program BIOGRAF (Molecular Simulations Waltham, Mass.).The topographic receptor map was energy-minimized to optimize contacts(Dreiding II forcefield (3)). Images were created with the programBIOGRAF (Molecular Simulations Waltham, Mass.). FIG. 1 depicts thetopographic receptor map Z thus generated to facilitate discernment ofthose structural features of the peptides that correlate with effects ofmemory retention. The Dreiding structural representation permits muchbetter visualization of the superimposition of the peptides on Z than dospace-filling CPK models.

Numbering clockwise from the bottom left on FIG. 1, No. 1, thepostulated sites on Z are characterized as (1) H-bonding; (2) aromatic;(3) cationic; (4) aromatic; and (5) anionic. Three views of the proposedsurface of Z are shown (FIGS. 1-3). FIGS. 4 through 18 illustrate thefit to Z of relevant peptides in order of their discussion in thisspecification.

Attachment of Val Phe Phe to Z (FIGS. 4-6) is presumed to take place byinteraction of the α-amino group of the N-terminal Val with anionic site5 and, by association of the Phe-1 and Phe-2 residues with aromaticsites 4 and 2, respectively. The designations Phe-1 and Phe-2 are givento the Phe residues in a particular peptide in the order of theoccurrence from the N-terminus.

There are five potential H-bonding sites on the side-chains of Val PhePhe, one each on the two oxygen atoms of the C-terminal carboxyl groupand three on the N-terminal amino group, for a total of five groups permolecule that may readily H-bond with water. Bound water molecules, inturn, may form H-bonds with other water molecules, in this mannerorganizing water clusters the sizes and shapes of which around theH-bonding groups are determined by combinatorial influences of molecularand environmental factors. Unbound Z site is presumed to have waterclusters associated with sites 1, 3 and 5 (FIG. 1). Val Phe Phe (FIG. 4)is posited to orient on Z in such a manner that the attractive energybetween Val Phe Phe and Z becomes great enough to squeeze outintervening water, in this way minimizing the energy of the system. Inother words, the attractive forces between Z and Val Phe Phe aresufficiently greater than those of either one of them for water thatthey associate preferentially with each other, releasing bound water inthe process.

The molar amnestic efficacy of Val Phe Phe was not significantlydifferent from that of the β-A4 homologue [Gln¹¹ ]β-(1-28) (FIG. 20A).If binding of its Val Phe Phe segment to Z is key to the amnestic effectof the latter, the flanking regions must contribute to strength ofattachment since neither the α-amino group of V nor the carboxyl groupof Phe-2 is available to contribute to binding efficacy.

If residues with H-bonding side-chains that do not bind to Z are addedat either end of a peptide that, by itself, binds to Z, water clusterson the non-binding moieties may become sufficiently large and stable sothat resulting physical and energetic barriers deter effectiveattachment of the peptide to Z. A curvilinearly decremental trend inamnestic potency was noted with increasing numbers of side-chainH-bonding groups in such peptides related to Val Phe Phe (FIG. 19, curveA).

Phe Phe Val, which also is amnestic (FIG. 19, curve B) shares aromaticsites 2 and 4 on Z with Val Phe Phe, but the C-terminal carboxyl groupof Val in Phe Phe Val is directed to cationic site 3 (FIG. 1, No. 7).Addition of binding-irrelevant, H-bonding groups to Phe Phe Val alsoreduced amnestic potency (FIG. 19, curve B). It is presumed that a watercluster can be mobilized on the amide nitrogen of a peptide bond whenGly is the C-terminal residue in the peptide because, uniquely among theamino acids, C-terminal Gly offers no side-chain interference to watercluster formation. For the latter reason, the number of H-bonding groupsassigned to a peptide was increased by one in the case of peptides witha C-terminal Gly (FIG. 2B; Phe Phe Val Gly (SEQ ID NO:11) and Asp PhePhe Val Gly).

Lys Leu Val Phe Phe Ala Glu (SEQ ID NO: 13), which comprises residues16-22 of β-A4, was not amnestic (FIG. 19, curve A), while the inversionof Val Phe Phe to give Lys Leu Phe Phe Val Ala Glu (SEQ ID NO:9)resulted in an amnestic peptide (FIG. 2, upper right; FIG. 20C). Lys LeuVal Phe Phe Ala Glu (SEQ ID NO: 13) fits site Z well, making 5-pointattachment in the following manner (FIG. 10): site 1, α-amino group ofLys; site 2, Phe-1; site 3, α-carboxyl group of Glu; site 4, Phe-2; site5, ε-amino group of Lys. If the Phe-1 and Phe-2 residues of SEQ ID NO:13 are arranged so as to fit sites 2 and 4, respectively, the α-carboxylgroup of Glu could interact coulombically with cationic site 3 (FIG. 1,Nos. 8 and 9); but the unrealistic energetic demand of the naked aqueousexposure of the hydrophobic Leu Val Phe segment that arises in thisconfiguration (FIGS. 8 and 9) would preclude the existence of such aconformation of the peptide in this instance and, therefore, its bindingto Z.

Although SEQ ID NO: 13 is not amnestic, Val Phe Phe Ala Glu is amnestic(FIG. 19, curve A). The latter peptide attaches in the following manner(figure not shown): site 5, α-amino group of Val; site 4, Phe-1; site 2,Phe-2; and site 3, α-carboxyl group of Glu. The lesser potency of ValPhe Phe Ala Glu than that of Val Phe Phe may be attributable to thegreater energy required for desolvation of Val Phe Phe Ala Glu to takeplace (FIG. 19, curve A).

Peptide β-(1-11) (SEQ ID NO:6), which does not have a Val Phe Phesequence, is not amnestic (trials to criterion 6.93±0.28 compared to6.80±0.21 for vehicle controls). Assuming that β-A4 binds to the sitethat Z was designed to represent, the latter and other observationsuggest the possibility that β-A4 and some of its larger amnesticfragments, e.g., β-(12-28) (SEQ ID NO:2), may require for attachment ona small segment that includes the Val Phe Phe sequence or a portionthereof. That even a small peptidic segment of a large protein moleculemay have great functional significance is illustrated by the fact thatthe tripeptide, Arg Gly Asp (SEQ ID NO: 14), is integral to the cellattachment site of fibronectin and may be involved in cellattachment-promoting activity of collagen as well as of a variety ofother proteins of great functional importance (4). In this regard, it isof interest that single or multiple Val Phe Phe sequences exist inapproximately 3% of the protein sequences in the Swiss-Prot data base.Among the Val Phe Phe-containing proteins are receptors for serotonin,acetylcholine, norepinephrine, γ-aminobutyric acid, glucocorticoids,androgens, progesterone, natriuretic peptide, insulin, and transferrin.Aberrant binding of β-A4 or related substances to Val Phe Phe bidingsites on membranes may wreak havoc with cellular signal transduction.β-A4 and the larger amnestic fragments derived from it possibly maycombine with Z in the form of aggregates rather than as monomers. Suchpeptides are known to self-associate, forming anti-parallel β-sheets ofstill undetermined structure (5-9). β-A4 also binds with high avidity toapolipoprotein Glu, with which it coexists in senile plaques; and thiscomplex might bind to Z (10, 11). Both Phe residues in Val Phe Phe arenot required to produce amnesia, since Val Phe (FIG. 1, No. 11) and PheVal (FIG. 1, No. 12) also were amnestic. However, neither Phe Phe (FIG.1, No. 13) nor Val Val (not shown) was amnestic (trials to criterion:vehicle, 6.80±0.21; Val Phe, 9.13±0.42, p<0.01; Phe Val, 0.4±0.46,p<0.01; Phe Phe, 7.0±0.42, ns; Val Val, 6.8±0.31, ns). Val Phe and PheVal both fit well to portions of Z, their Phe groups binding to aromaticsite 4, the free amino group of Val binding to anionic site 5 and thefree carboxyl group of Val in Phe Val binding to cationic site 3.

The best fit of Phe Phe to Z probably would occur if Phe-1 and Phe-2were apposed to aromatic sites 4 and 2, respectively (FIGS. 13 and 14).However, this would require aqueous exposure of the closely groupedhydrophilic N-terminal amino group, the C-terminal carboxyl group, andthe peptidic amide group. The energy required to remove resultant waterclusters from Phe Phe for binding to take place in this form probablywould be too great to permit its effective binding of Phe Phe to Z. IfVal Val were oriented on Z in a manner similar to Val Phe, its affinityfor Z would be less than that of Val Phe because the energy of bindingof the C-terminal Val to aromatic site 4 would be considerably less thanthat of Phe. The probability of attachment of Val Val to the twoaromatic sites of Z would be less even than that of Phe Phe.

Vasoactive intestinal peptide (VIP) (His Ser Asp Ala Val Phe Thr Asp AsnTyr Thr Arg Leu Lys Gln Met Ala Val Lys Lys Tyr Leu Asn Ser Ile LeuAsn-NH₂ (SEQ ID NO: 15)) is amnestic in the FAAT paradigm (12). Ala ValPhe Thr (FIG. 16), comprising residues 4-7 (underscored above) of this27-residue peptide, was amnestic with a molar efficacy similar to thatof VIP (FIG. 3B). Val Phe Thr (residue 5-7) also was amnestic (FIG. 15;FIG. 3B), but Ala Val Phe (residue 4-6; FIG. 1, No. 17) was not (FIG.3B). Val Phe Thr fits excellently to a portion of Z, with Val and Thrinteracting coulombically with sites 5 and 3 through their respectivefree amino and free carboxy groups, with Phe associating with aromaticsite 4. Although the Ala residue of Ala Val Phe Thr (FIG. 16) protrudesfrom the surface of Z into the aqueous medium, the disposition of thehydrophobic portions of Ala and Val is such as to tend to preventformation of large water clusters around the closely lying hydrophilicgroups. Ala Val Phe would bind to Z poorly, at best, because the mostfavorable binding sites for the uncharged internal Val residue and forthe Phe would be aromatic sites 2 and 4, respectively, the affinity ofAla Val Phe for Z probably lying midway between that of Val Val and PhePhe, which are amnestically ineffective (see above).

The 2-Dimensional Model. A 2-dimensional model of Z was constructed fromconsideration of data discussed in the preceding section and wasinfluenced by the proposed 5-site interaction with Lys Leu Phe Phe ValAla Glu (SEQ ID NO: 9) (FIG. 10). Every amnestic peptide tested up tothat point contained a Val residue, and the small amnestic peptides werefragments of larger ones (β-A4 and VIP) that also were amnestic. It wasrecognized that credibility of the model would be enhanced if a Val-freeamnestic fragment of a memory-enhancing peptide were to be found to fitthe topography of Z.

A likely candidate for the later was the tetrapeptide Gln Phe Phe Gly(SEQ ID NO: 12) (underscored below) (FIG. 18) that comprises residues6-9 of the 11 residue substance Pro (Pro Arg Pro Gly Pro Gln Gln Phe PheGly Leu Met-NH₂ (SEQ ID NO: 16)). Substance Pro has been reported toenhance memory retention (e.g., see references 13-16) and to counteracttrophic and toxic effects of B-A4 on hippocampal neurons in culture (seereference 17). Gln Phe Phe Gly fits the original Z surface remarkablywell at sites 1, 2, 4 and 5. The substitution of γ-aminobutyric acid forGly at the C-terminus improved the fit to site 3, the latter substance(SEQ ID NO:20) being used to devise the current receptor map (FIGS.1-3).

Referring to FIG. 18, the γ-amide group of the Gln residue of Gln PhePhe Gly (SEQ ID NO: 12) H-bonds to site 1 and its α-amino groupinteracts electrostatically with site 5. Residues Phe-1 and Phe-2associate with aromatic sites 4 and 2, respectively, and the carboxylgroup of the C-terminal Gly falls just close enough to site 3 to engagein weak coulombic interaction with it. With γ-aminobutyric acidsubstituting for Gly, the terminal carboxyl group fits perfectly ontosite 3.

The fact that Gln Phe Phe Gly (SEQ ID NO: 12), a Val-free fragment ofthe memory-enhancing substance Pro, is itself amnestic accords with thepremise that Z defines the generally features of a binding site foramnestic substances. Structural requirements for binding to Z appear tobe quite fastidious since some peptides similar to Gln Phe Phe Gly arenot amnestic. The mean trials to criterion ±SEM and p values forcomparison with vehicle are: vehicle alone, 6.80±0.21, Gln Phe Phe Gly,0.0±0.46, p<0.01; Gln Phe Val Gly, 6.80±0.39, ns; Ser Phe Phe Gly,7.33±0.46, ns; Ser Phe Val Gly 7.47±0.47, ns. The model Z is the basisfor the devisal of memory-enhancing substances.

Dual effects usually are observed in studies of memory enhancement byexitatory substances (18). In the effective dose ranges, progressivelyincreasing doses first increase response to a maximum, beyond whichdecreasing responses are observed, until a dose is reached at which nosignificant effects are seen over the controls. A copendium of resultsin the literature showed that this phenomenon was observed for 27memory-enhancing compounds given by seven routes of administration tosix different species of organisms in 15 laboratories (19).

The model Z was developed using an amnestic paradigm. Actually, six keysubstances used in the development of Z are memory-enhancers at farlower doses than those at which they show amnestic effects (FIGS. 22-24and Table 1). The groups on Z with which these substances are presumedto combine are shown in Table 1.

Val Phe Phe is a memory enhancer in weakly trained animals (FIG. 21A)and amnestic when tested in much higher amounts in well-trained animals(FIG. 21B) Statistically significant memory enhancement was first foundat 1.9×10⁻¹¹ moles/mouse and amnesia first noted at 3×10⁻⁹ moles/mouse.thus, memory enhancement was noted first at a dose less thanone-hundredth (0.63%) of that which first produced amnesia. As notedpreviously in the discussion of the model, Val Phe Phe is proposed tocombine with Z at three points of attachment one coulombic and two ofthem with aromatic groups (Table 1). Comparison of Val Phe Phe to memoryenhancement with Val Val Phe and Phe Phe Val Gly showed them to haveclosely similar potencies (FIG. 5A-C).

Both Val Phe and Phe Val gave closely similar memory-enhancing results(FIGS. 23A and 23B), showing approximately the same potency as Val PhePhe (Table 1).

All of the above proved to be much weaker than Gln Phe Phe Gly (Table1). Gln Phe Phe Gly is by far the most potent, producing statisticallysignificant memory enhancement at 10⁻¹⁴ moles/mouse.

                                      TABLE 1    __________________________________________________________________________    Binding Characteristics on Z of 5 Memory-Enhancing Peptides    Groups on Z to                Lowest Concentration For Significant    Which the Peptides Bind (See FIGS. 1-3)                                  Memory Enhancement    Peptides         1     2    3    4    5          P Values For Comparison    Tested         H-Bonding               Aromatic                    Cationic                         Aromatic                              Anionic                                  Moles/Mouse                                         With Vehicle    __________________________________________________________________________    Val Phe         -     +    -    +    +   1.9 × 10.sup.-11                                         <0.01    Phe    Val Phe         -     -    +    +    +   2 × 10.sup.-11                                         <0.05    Phe Val         -     -    +    +    +   2 × 10.sup.-11                                         <0.05    Val Val         -     +    -    +    +   1 × 10.sup.-11                                         <0.05    Phe    Phe Phe         -     +    +    +    -   2 × 10.sup.-11                                         <0.05    Val Gly    Gln Phe         +     +    +    +    +   6 × 10.sup.-14                                         <0.05    Phe Gly    __________________________________________________________________________

Additional Peptides and Non-peptidic Substances as Memory Enhancers.

Structure-activity considerations suggest several substances mentionedbelow that may be effective memory enhancers in the same manner as theeffective peptides thus far discovered.

Since by far the most effective memory-enhancing peptide discovered todate by the method developed above is Gln Phe Phe Gly (SEQ ID NO: 12),which touches at all five sites of the postulated memory-related bindingsites (Table 1), only those peptides which do likewise are consideredbelow. Computer-assisted fitting procedures and manual work with CPKspace-filling molecular models has revealed a series of peptides thatfit to Z even more exactly than does Gln Phe Phe Gly. These arediscussed below.

Memory-enhancing Peptides Based on Postulated Interaction with All FiveSites on Z.

1. Compounds related to Gln Phe Phe Thr (SEQ ID NO: 17)--C-terminalsubstitution

(a) Gln Phe Phe-γ-aminobutyric acid (SEQ ID NO:20)

(b) Gln Phe Phe-β-alanine (SEQ ID NO:21)

(c) Gln Phe Phe-δ-aminovaleric acid (SEQ ID NO:22)

Substances (a)-(c) above fit with their C-terminal carboxyl groupscloser to cationic site 3 on Z than does Gln Phe Phe Gly and areexpected to be more potent memory enhancers than Gln Phe Phe Gly.

2. Compounds related to Gln Phe Phe Thr (SEQ ID NO: 17)--N-terminalsubstitution

(a) Lys Phe Phe Gly (SEQ ID NO: 18)

(b) Arg Phe Phe Gly (SEQ ID NO: 19)

(c) homoarginyl Phe Phe Gly (SEQ ID NO:23)

(d) ornithyl Phe Phe Gly (SEQ ID NO:24)

The substitution of Lys and other aliphatic basic amino acids for Gln inGln Phe Phe Gly is based on the observation that in Lys Leu Phe Phe ValAla Glu (SEQ ID NO: 9), a potent amnestic (FIG. 1, No. 10), the ε-aminogroup of Lys can bind to the H-bonding group of site 1 on Z. If this beso, H-bonding via the δ-amino group of ornithine or the guanidino groupof Arg or homoarginine also can take place.

3. Compounds related to Gln Phe Phe Gly-substitution for both Gln andGly with compounds selected from 1a-c and 2a-d while keeping Phe Pheconstant, e.g., Arg Phe Phe-δ-aminobutyric acid.

All told, twelve such combinations are possible, in addition to sevenlisted under items 1 and 2 above.

4. Compounds related to Gln Phe Phe Gly-substitution for Phe-1 and/orPhe-2 by His and/or Tyr in Gln Phe Phe Thr and all of the substanceslisted in 1a-c and 2a-d.

His and Tyr have properties sufficiently similar to those of Phe so thattheir affinities for aromatic sites 2 and 4 on Z might be equal to orgreater than that of Phe. Arg His Tyr-δ-aminobutyric acid, Lys TyrTry-β-alanine, etc. This gives a total of 160 compounds that can betested in this series.

The grand total of 179 substances listed in items 1-4 above does notinclude many possible variations of the N-terminal or C-terminal groupsin the above group of tetrapeptides, such a acetylation of the former oresterification of the latter.

Memory-enhancing Non-peptide Substances Based on Postulated Interactionwith All Five Sites on Z.

A long structural search has yielded a new semi-rigid compound (seeFIG. 1) that fits Z at all postulated sites at least as well as any ofthe above peptides. ##STR1##

FORMULA I

Providing the ring structure remains intact, many substitutions arepossible in the above, e.g., substitutions on the benzene rings,substitution of the latter by imidazole groups, substitution of halogens(fluorine or chlorine) for the carboxypropyl group facing site 3, etc.The compound shown above is only one example of how the Z model can beused in design of potential memory-enhancing substances.

References

1. Flood, J. F., et al. Proc. Natl. Acad. Sci. USA 88:3363-3366 (1991)

2. Connolly, M. L., Science 221:709-713 (1983)

3. Mayo, S. L., et al. J. Phys. Chem. 94:8897-8909 (1990)

4. Ruoslahti, E., Ann. Rev. Biochem. 57:375-413 (1988)

5. Kirschner, D. A., et al., Proc. Natl. Acad. Sci. USA 84:6953-6957(1987)

6. Maggio, J. E., et al. Proc. Natl. Acad. Sci. USA 89:5462-5466 (1992)

7. Hilbich, C., et al., J. Mol. Biol. 228:460-473 (1992)

8. Zagorski, M. G., Biochemistry 31:5621-5631 (1992)

9. Jarrett, J. T., Biochemistry 32:4693-4697 (1993)

10. Wisniewski, E., et al., Biochem. Biophys. Res. Commun. 192:359-365(1993)

11. Strittmatter, W. J., et al., Proc. Natl. Acad. Sci. USA 90:1977-1981(1993)

12. Flood, J. F., et al., Peptides 11:933-938 (1990)

13. Wetzel, W., et al., Acta biol. med. germ. 41:647-652 (1982)

14. Schlesinger, K., et al. Behav. Neural Biol. 45:230-239 (1986)

15. Hasenohrl, E. U., et al. Peptides 11:163-167 (1990)

16. Nagel, J. A., et al. Peptides 14:85-95 (1993)

17. Kosik, K., et al., Neurobiology of Aging 13:535-625 (Pergamon Press,New York) (1992)

18. Cherkin, A., et al., In: Cellular Mechanisms of Conditioning andBehavioral Plasticity (C. D. Woody, D. L. Alkon and J. L. McGaugh, eds.)Plenum Press, New York and London, pp. 343-354 (1988)

19. Drummond, G. I., Adv. Cyclic Nucleotide Res. 15:373-494 (1983)

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 24    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    AspAlaGluPheArgHisAspSerGlyTyrGlnValHisHisGlnLys    151015    LeuValPhePheAlaGluAspValGlySerAsnLys    2025    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 17 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    ValHisHisGlnLysLeuValPhePheAlaGluAspValGlySerAsn    151015    Lys    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    ValPhePheAlaGluAspValGlySerAsnLys    1510    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    ValHisHisGlnLysLeuValPhePhe    15    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    ValPhePhe    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 11 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    AspAlaGluPheArgHisAspSerGlyTyrGln    1510    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    AlaValPheThr    1    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    ValIlePro    1    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    LysLeuPhePheValAlaGlu    15    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 6 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    ValPhePheValValPhe    15    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    PhePheValGly    1    (2) INFORMATION FOR SEQ ID NO:12:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    GlnPhePheGly    1    (2) INFORMATION FOR SEQ ID NO:13:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 7 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    LysLeuValPhePheAlaGlu    15    (2) INFORMATION FOR SEQ ID NO:14:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    ArgGlyAsp    1    (2) INFORMATION FOR SEQ ID NO:15:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    HisSerAspAlaValPheThrAspAsnTyrThrArgLeuLysGlnMet    151015    AlaValLysLysTyrLeuAsnSerIleLeuAsn    2025    (2) INFORMATION FOR SEQ ID NO:16:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 12 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: N-terminal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    ProArgProGlyProGlnGlnPhePheGlyLeuMet    1510    (2) INFORMATION FOR SEQ ID NO:17:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    GlnPhePheThr    1    (2) INFORMATION FOR SEQ ID NO:18:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    LysPhePheGly    1    (2) INFORMATION FOR SEQ ID NO:19:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    ArgPhePheGly    1    (2) INFORMATION FOR SEQ ID NO:20:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 4    (D) OTHER INFORMATION: gamma aminobutyric acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    GlnPhePheXaa    1    (2) INFORMATION FOR SEQ ID NO:21:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 4    (D) OTHER INFORMATION: beta alanine    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    GlnPhePheXaa    1    (2) INFORMATION FOR SEQ ID NO:22:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 4    (D) OTHER INFORMATION: delta aminovaleric acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    GlnPhePheXaa    1    (2) INFORMATION FOR SEQ ID NO:23:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: homoarginine    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    XaaPhePheGly    1    (2) INFORMATION FOR SEQ ID NO:24:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: Not Relevant    (ii) MOLECULE TYPE: peptide    (iii) HYPOTHETICAL: YES    (v) FRAGMENT TYPE: internal    (ix) FEATURE:    (A) NAME/KEY: Modified-site    (B) LOCATION: 1    (D) OTHER INFORMATION: ornithine    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    XaaPhePheGly    1    __________________________________________________________________________

I claim:
 1. Each of the compounds Gln Phe Phe-γ-aminobutyric acid (SEQ ID NO: 20) Gln Phe Phe-β-alanine (SEQ ID NO: 21) Gln Phe Phe-δ-aminovaleric acid (SEQ ID NO:22) Lys Phe Phe Gly (SEQ ID NO: 18) Arg Phe Phe Gly (SEQ ID NO: 19) Homoarginyl Phe Phe Gly (SEQ ID NO: 23) Ornithyl Phe Phe Gly (SEQ ID NO: 24). 